The CUL4A ubiquitin ligase gene is frequently amplified or overexpressed in a wide variety of tumor types, including breast cancer, hepatocellular carcinomas, mesotheliomas, and squamous cell carcinomas. While recent studies have identified the components of the multimeric CUL4A E3 ligase complex and several cellular targets, the role of CUL4A in tumorigenesis remains largely elusive. We and others showed that CUL4A plays an inhibitory role in nucleotide excision repair through targeted degradation of the DDB2 and XPC DNA damage sensors, as well as the G1/S DNA damage checkpoint protein p21. Importantly, our skin-specific CUL4A knockout mice are hyper- resistant to UV- and chemical carcinogen-induced skin cancer, and are otherwise healthy and display no abnormalities, suggesting that CUL4A may be an attractive target for cancer prevention. The primary objective is to develop and optimize a high- throughput screening assay to identify small molecule inhibitors of the CUL4A ubiquitin ligase from chemical libraries at the NIH Chemical Genomic Center (NCGC). This application is built upon the extensive biochemical, cell biological, and genetic characterization of the CUL4A ubiquitin ligase performed in Dr. Pengbo Zhou's lab during the last 10 years, and the comprehensive array of in vitro and cell-based assays as well as the mouse knockout models to examine CUL4A-DDB1 functions. The studies are complemented by the extensive high-throughput screening expertise of Dr. Yueming Li's group at Memorial Sloan-Kettering Cancer and Dr. Wei Zheng's group at the NIH Chemical Genomic Center. We are uniquely positioned to pursue the following two specific aims: (1) to develop and optimize an AlphaLISA(R)-based HTS assay for CUL4A- DDB1 interaction;(2) to configure secondary HTS assays required to evaluate the hit compounds. Successful completion of the proposed studies will set the stage for application of the CUL4A-DDB1(BPB) AlphaLISA(R) assay on the HTS platform to screen the NCGC 300K chemical libraries and identify small molecule inhibitors of the CUL4A- DDB1 ubiquitin ligase. These studies represent the first step towards evaluating the efficacy of pharmacological CUL4A inhibition as an effective approach for cancer prevention and intervention. PUBLIC HEALTH RELEVANCE: The CUL4A gene is frequently found amplified or overexpressed in a wide variety of tumor types, including breast cancer, hepatocellulat carcinomas, mesotheliomas and squamous cell carcinomas. Recent studies suggest that abrogation of CUL4A presents significant benefits in enhancing DNA repair and DNA damage response, and protects against carcinogenesis. However, there is currently no pharmacological agent available for blocking the CUL4A ubiquitin ligase activity. Our overall goal is to develop pharmacological inhibitors against the DDB-CUL4A (or CRL4) ubiquitin ligase as anticancer agents. Here we propose to take the critical first step to develop and optimize a high throughput screening assay and to establish secondary validation assays, with the aim of implementing a high throughput screening of large chemical libraries to identify small molecule inhibitors of the CUL4A ubiquitin ligase. This R21 project expected to set the stage for developing pharmacological agents that selectively target CUL4A as novel cancer prevention and/or therapeutic agents.